The broad, long-term objectives of this research are to elucidate the mechanisms underlying the increase in insulin sensitivity for glucose metabolism that is a hallmark and major health benefit of calorie restriction (CR; consuming 60% of ad libitum, AL, intake) and to understand other important roles of insulin signaling for CR effects in old age. Our novel model is that CR induces tissue-specific and pathway-specific effects on insulin signaling. The model predicts that for CR vs. AL rats with endogenous insulin (basal or after intravenous, IV, glucose challenge), in vivo insulin signaling involved in glucoregulation will be similar or increased in classic target tissues (skeletal muscle, adipose, liver), but reduced in non-classic target tissues (brain, kidney, aorta) that are not major sites of insulin-stimulated glucose disposal. Enhanced insulin signaling for glucoregulatory processes of classic tissues would be a plausible explanation for increased whole body glucose clearance despite much lower insulin with CR. The model also predicts that insulin signaling pathways that are not implicated in glucoregulation will have lower activation in vivo for CR vs. AL rats in both classic and non-classic tissues. Aim 1 will elucidate mechanisms leading to improved insulin action induced by CR (begun at ~3.5mo-old) in Adult (12mo) and Old (25mo) rats. Identifying the specific signaling steps in muscle with CR is important because by age 60-74yr, ~1/3 of Americans suffer from abnormal glucose tolerance, and muscle insulin resistance is an essential defect in age-related progression to type 2 diabetes. In addition to insulin's central role in glucoregulation, compelling evidence in primitive organisms (yeast, nematodes and flies) points to the insulin signaling pathway as a key modulator of primary aging. However, knowledge about the influence of long-term CR on in vivo insulin signaling in mammalsis remarkably limited. Therefore, Aims 2 (IV glucose challenge) and 3 (IV insulin challenge) will ascertain the effects of CR and age on in vivo insulin signaling pathways either with or without glucoregulatory roles in multiple tissues (both classic and non-classic) of Adult and Old rats. We expect an IV insulin challenge to induce in CR vs. AL (regardless of age) greater insulin-signaling related to glucoregulation in classic, but not in non-classic target tissues. The results of these studies will provide novel insights into the effects of CR and age on insulin signaling that may be valuable for developing interventions to oppose age-related deficits.